Cdon Acts As a Hedgehog Decoy Receptor During Proximal-Distal Patterning of the Optic Vesicle
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ARTICLE Received 10 Dec 2013 | Accepted 26 May 2014 | Published 8 Jul 2014 DOI: 10.1038/ncomms5272 OPEN Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle Marcos Julia´n Cardozo1,2, Luisa Sa´nchez-Arrones1,2,A´frica Sandonis1,2, Cristina Sa´nchez-Camacho2,w, Gaia Gestri3, Stephen W. Wilson3, Isabel Guerrero1 & Paola Bovolenta1,2 Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex with the Hh receptor Patched 1 (Ptc1). This receptor complex binds Hh and enhances signalling activation, indicating that Cdon positively regulates the pathway. Here we show that in the developing zebrafish and chick optic vesicle, in which cdon and ptc1 are expressed with a complementary pattern, Cdon acts as a negative Hh signalling regulator. Cdon predominantly localizes to the basolateral side of neuroepithelial cells, promotes the enlargement of the neuroepithelial basal end-foot and traps Hh protein, thereby limiting its dispersion. This Ptc-independent function protects the retinal primordium from Hh activity, defines the stalk/retina boundary and thus the correct proximo-distal patterning of the eye. 1 Centro de Biologı´a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı´ficas—Universidad Auto´noma de Madrid, 28049 Madrid Spain. 2 Ciber de Enfermedades Raras (CIBERER), ISCIII, c/ Nicolas Cabrera 1, 28049 Madrid Spain. 3 Department of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. w Present address: Universidad Europea, C/ Tajo, s/n Villaviciosa de Odo´n, 28670 Madrid, Spain. Correspondence and requests for materials should be addressed to P.B. (email: [email protected]). NATURE COMMUNICATIONS | 5:4272 | DOI: 10.1038/ncomms5272 | www.nature.com/naturecommunications 1 & 2014 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5272 he Hedgehog (Hh) signalling pathway is a highly expression, whereas ptc1 matches Pax2 distribution26,34,35 conserved regulator of patterning and homeostatic events (Fig. 1f–i; Supplementary Fig. 1). Here we have addressed Tin various developing and adult organs and thus its whether and how cdon contributes to optic vesicle patterning, activation is under tight control1,2. Hh ligands are released using zebrafish and chick embryos as model systems. Our results through the cooperative activity of the transmembrane protein indicate that in the eye, Cdon acts as a negative regulator of Hh Dispatched and heparan sulphate proteoglycans, which are also signalling. Cdon as well as Boc expand the surface of the basal crucial to modulate long-range distribution3–5. There is also end-foot of neuroepithelial cells, bind Hh and prevent its evidence that cytonemes, long filopodial-like extensions formed dispersion, thus limiting long-range signalling, as proposed for by the producing cells, promote delivery of Hh-containing Ihog and Boi19,36–38. This function protects the retinal vesicles to similar extensions in target cells6–8. In the targets, primordium from Hh activity, thus enabling a correct P-D binding of Hh to a receptor complex containing the seven-pass patterning of the eye. membrane protein Patched (Ptc), releases Ptc-mediated inhibition of the signal transducer protein Smoothened (Smo), Results which initiates a cascade of intracellular events that lead to the 9,10 cdon and ptc localize to complementary optic vesicle domains. transcription of Hh target genes . Pax2 function is critical to establish optic stalk identity in dif- Cdon (cell adhesion molecule-related, downregulated by 32,33,39 11,12 13 ferent vertebrate species and to restrain Pax6 expression, so oncogenes) , the related Boc (Brother of Cdo) or their that the two genes have a complementary expression restricted to Drosophila counterparts Ihog (Interference hedgehog) and Boi the proximal/stalk and distal/retinal optic vesicle, respectively29,32 (Brother of Ihog), are additional components of the Hh receptor (Supplementary Fig. 1). Pax2 expression is regulated by Hh, complex14. Cdon and Boc are cell surface glycoproteins that which is strongly expressed in the ventral midline30,32,33. To get a belong to a subgroup of the immunoglobulin (Ig) super-family of better understanding of the role of Hh signalling in the P-D cell adhesion molecules14. Their ectodomains contain five and patterning of the eye, we analysed the distribution of Hh in four immunoglobulin-like domains, respectively, followed by zebrafish embryos. Hh was strongly localized to cells of the three fibronectin III (FNIII) repeats (1–3), which are involved in 15 ventral midline of the forebrain and in optic stalk cells (Fig. 1a–e). Ptc and Hh binding. FnIII(1-2) domains interact with Ptc , At early stages of optic vesicle specification, Hh strongly whereas the evolutionary conserved FnIII(3) domain binds Hh 16–18 accumulated at the most ventrally positioned Pax2-positive cells with high affinity . (Fig. 1c) but its distribution extended dorsolaterally as develop- The requirement of a heteromeric interaction between Ptc and ment progressed (Fig. 1d,e). In agreement with this distribution, Ihog/Boi for both high-affinity Hh binding and presentation of 19 the Hh receptor ptc1 was expressed in the ventral forebrain Ptc at the cell surface was initially demonstrated in Drosophila 34,40 15 including the prospective optic stalk (Fig. 1f,g). In contrast, and then shown to be conserved in vertebrates .Inmice, cdon, but not the related boc28 (Supplementary Fig. 1w–z), was Cdon and Boc and the unrelated Hh binding protein Gas1 strongly localized to the neural retina region (Fig. 1h,i). This (refs 20,21) act as positive and redundant regulators of Hh distribution was conserved in chick and mouse embryos signalling in different contexts22–24.Forexample,Cdon-deficient (Supplementary Fig. 1k–o). In the latter, Cdon protein appeared mice are characterized by mild holoprosencephalia (HPE) with to accumulate at the basolateral membrane of the retinal microphthalmia and moderate facial and brain defects of strain 11,25–27 epithelium starting from E10, resembling the subcellular specific penetrance . These traits are aggravated by the 4,36 À / À À / À localization described for the Drosophila homologue Ihog additional inactivation of Boc,sothatCdon ;Boc embryos (Supplementary Fig. 1t–v). present a significant loss of the Hh pathway activity24,whichis further worsened by Gas1 inactivation. Indeed Cdon À / À ; Boc À / À ;Gas1 À / À compound mutant embryos show a cdon knockdown alters P-D patterning of the eye primordium. phenotype similar to that caused by the loss of Sonic hedgehog The complementary pattern of ptc1 and cdon expression was (Shh) function: strong facial malformations and failure of partition consistent with the idea that, in the eye, the two proteins could act of the cerebral hemispheres and eye primordia22.Inzebrafish, independently without forming a heteromeric Hh receptor15, analysis of the uml(boc) mutant supports the idea that Boc acts as a raising the question of whether and how cdon expression in the positive regulator of Hh signalling in the ventral neural tube but retina contributes to eye development. Answering these might have an opposite function in the lower jaw28. questions, however, requires local or partial interference with In vertebrates, the morphogenesis of the eye primordium, also Cdon function because shh colocalizes with cdon at the axial known as optic vesicle, occurs concomitant to its patterning midline of gastrulating embryos (Fig. 1f,g). To avoid the complete along the proximal-distal (P-D) axis in medial (prospective optic abrogation of this early function that may be responsible for the stalk) and lateral (prospective retinal) domains. The establish- mild holoprosencephalic phenotype of Cdon null mouse ment of this early P-D patterning is defined by the expression of embryos11, we reduced cdon expression levels in zebrafish two pair- and homeobox- containing transcription factors, Pax2 embryos using specific morpholinos (MOs). and Pax6, the expression of which is, respectively, restricted to the Doses of 160 mM of a MO that efficiently interfered with the optic stalk and the retinal primordium29–31. Functional studies cdon translation start site (CdonATG MO; Supplementary Fig. 2a–e) have demonstrated that Hh secreted from the midline of the generated morphants, which were characterized by abnormal ventral forebrain is required to promote Pax2 expression and folding of the retinal ventronasal quadrant and the presence of an thus to establish optic stalk identity30,32,33. A cross-repressing optic fissure coloboma (lack of fissure fusion; Supplementary regulatory loop between Pax2 and Pax6 then refines the pattern, Fig. 2f–i; higher doses led to early lethality), often a consequence of forming a precise boundary between the retina and the optic abnormal optic stalk development31,41,42. The trunk in morphants, stalk29. In the absence of Hh signalling, the stalk does not form however, appeared normal with no apparent ventral midline and Pax6-positive vesicles do not separate, forming a single defects suggesting that cdon knockdown was compatible with axial cyclopic eye33. midline formation. These anterior defects were not found in Notably, in the